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Background

In anatomical and surgical pathology, the customary method of manual observation and
measurement of immunohistochemically stained markers from microscopic images is tedious,
expensive and time consuming. There is great demand for automated procedures for analyzing
digital images (DIs) of these markers [1] given that they reduce human variability in the evaluation of stained markers [2,3] and increase the speed and efficiency of the analysis [4]. Computerized DI analysis software generally involves a stained objects/nuclei segmentation
method to detect and quantify the number of positively stained markers in combination
with the standard evaluation of their morphometric and/or densitometric features [5,6]. However, automatic segmentation often fails due to the presence of spurious stain
deposits in tissue sections (background). The “removal” of the background from noisy
DIs, so that only the objects of interest are identified, is difficult due to the
color values of pixels in the nuclei and background overlapping during the color segmentation
processes.

We previously developed an automated macro that allows quantification of several nuclear
markers in various neoplasic tissues [7]. In an attempt to standardize the immunohistochemical analysis and to improve cell
detection, we propose a new procedure that quantifies only positively stained nuclei
even though they have a similar color to that of the surrounding tissue. The aim of
this work was to develop a single automated procedure that allows images to be analyzed
irrespective of whether the spurious stain deposit in background is present or absent.
The multistep process includes algorithms that permit this discrimination so that
the appropriate procedure for optimal quantification can then be applied.

Materials and methods

Images

Histological sections of lymphomas and breast cancer tissues, previously immunohistochemically
stained with standardized protocols [8,9], were selected from the archives of the Department of Pathology of the Hospital de
Tortosa Verge de la Cinta, Catalonia, Spain. Staining was performed with monoclonal
antibodies directed against the nuclear protein estrogen receptors (ERs; clone NCL-ER-6F11,
Novocastra, Newcastle upon Tyne, UK), progesterone receptors (PRs; NCL-PGR-312, Novocastra),
Ki-67 (clone MIB-1, Dako, Carpinteria, CA, USA) and FOXP3 (clone FOXP3-236A/E7, CNIO,
Spain). The entire process was standardized to ensure high reproducibility and brown
staining homogeneity, which are very important requirements for image analysis [10]. This study received institutional review board approval.

Image capture

Stained tissue sections were viewed using brightfield illumination under a Leica DM
LB2 upright light microscope (Leica Microsystems Wetzlar GmbH, Wetzlar, Germany) with
a 40x plane-apochromatic objective. One hundred digital images were captured with
a Leica DFC320 digital camera connected to a computer and controlled with the Leica
IM50 v4.0 program. TIFF format DIs, with a resolution of 1392 x 1040 pixels (1.4 Mpixels)
in RGB 24 true-color format, were selected on the basis of the presence or absence
of the spurious stain deposits of the background, otherwise ensuring a variety of
concentrations and distributions of stained nuclei (Figure 1).

Figure 1. Illustration of original digital images of immunohistochemically stained nuclear markers
with different background level (A, B) and without background (C, D).

Procedure developed in the new procedure

The new automated multistep DI analysis procedure was developed with Image-Pro® Plus
5.0 software (Media Cybernetic, Silver Spring, USA). We had previously developed an
automated macro to quantify stained nuclei in images without background using an RGB
color model and iterative morphological segmentation [7]. This macro makes use of a wide color range to detect positive nuclei from the darkest
to the lightest positive brown color pixel and applies a mask to displace the pixel
color values of negative objects outside the segmentation color range of the positive
nuclei. However, in DIs with a background this macro does not segment DIs correctly
due to the similar color values of the positive nuclei and the background (Figure
2).

Figure 2. Example of active contour segmentation in digital images with positively stained nuclear
markers. Evident differences in the contours obtained with the old macro (A) and those
obtained with the new procedure (B).

We therefore developed a multistep procedure that discriminates DIs as a function
of the presence or absence of background and that enables the more appropriate of
the three macros to be applied directly. First, a mask overlaps objects with negative
and light positive-intensity pixels (Figure 3a) so that only positive objects with the darkest range of color (objects map 1) can
be selected (Figure 3b). Then, using a mask only for negative objects, the next two steps select the clearer
positive objects (objects map 2 and 3) using different color ranges and morphological
ranges of area and roundness (Figure 3c). The objects map 4 is the sum of all the positive objects in the three previous
steps. At the end of the positive selection, another step with a discriminative algorithm
is applied in which the non-selected brown color is segmented with two different ranges
of brown (Figure 3d). If no brown color is detected with the second range (area 2 = 0), the algorithm
determines that there is no background in DIs that are automatically analyzed with
the old macro (Figure 3e). On the other hand, when DIs with background were detected (area 2 > 0), they are
submitted to another algorithm that calculate the ratio of the area of the two color
ranges (Figure 3f). DIs with a low-level background (low ratio) are automatically analyzed with a restrictive
macro with the first three steps of the new procedure (Figure 3g). DIs with a high background level (high ratio) are automatically analyzed with the
new procedure, supplemented with a final step that selects the clearest objects using
color and morphological segmentation (Figure 3h). The three macros detect only positive objects (individual nuclei and clusters).
Information about the area of the positive objects detected with these macros is automatically
exported to an Excel datasheet (Figure 3i) containing several algorithms so that the number of definitive positive nuclei in
those selected objects can be estimated, as previously described [7].

Figure 3. Schematic representation of the automated multistep procedure. Image analysis was
carried out with the Image-Pro® Plus 5.0 program and data from selected positive objects
are exported to an Excel datasheet in which appropriate algorithms are implemented,
enabling the definitive number of positive cells to be calculated.

Quantification and statistical analysis

The mean of two manual counts made by two trained observers was taken as the reference
value (gold standard) to validate the results obtained with the old macro and the
newly proposed procedure. The comparisons made were: manual 1 versus manual 2 readings;
mean of manual readings versus old macro reading; mean of manual readings versus new
procedure reading; and the old macro versus the new procedure readings. The extents
of agreement between the manual and automatic results were evaluated with Bland-Altman
and Kaplan-Meier analyses with their corresponding graphs. Bland-Altman graphs illustrate
the differences between the compared methods with respect to the mean of each paired
count. Kaplan-Meier curves portray the conditional probability of observing differences
between results obtained from the methods compared. All statistical analyses were
carried out with SPSS 19.0.

Results and discussion

Overall, the Bland-Altman graph (Figure 4) indicates that count differences obtained comparing the new procedure vs. the mean
of the manual counts (green circle) were lower than the old macro vs. the mean of
the manual counts (red circle). Nevertheless, greater accuracy was obtained from images
with background (Figure 5A1) where the probability of observing a difference of 50 nuclei between the new procedure
and the manual quantification (22.7%, green curve) is less than half that between
the old macro and manual quantification (47.4%; red curve).

Figure 4. Bland-Altman graph showing differences between the two manual quantification methods
(in black), the mean of the manual counts and the old macro results (in red) and the
mean of the manual counts vs. the new procedure results (in green). The Y axis represents
the difference between the results from pairs of methods, and the X axis represents
the mean of both counts for each comparison.

Figure 5. Superimposed Kaplan-Meier curves comparing the probability of difference between the
manual reading 1 vs. manual reading 2 (in black), the mean of manual readings vs.
old macro (in red) and mean of manual readings vs. new procedure (in green). The X
axis represents count differences between the two methods or readings compared and
the Y axis represents the probability of observing these differences. The upper row
(A) represents the results obtained with DIs grouped in function of the background
level: DIs with background (A1) versus DIs without background (A2). The lower row
represents the results obtained with DIs grouped in function of their complexity:
high-complexity DIs (B1) and low-complexity DIs (B2).

As previously demonstrated, image complexity relative to the number of positively
stained nuclei may affect the automated nuclear quantification [8]. In the present study, DIs were divided as before into a low-complexity group (≤100
positively stained nuclei/images) and a high-complexity group (>100 positively stained
nuclei/image). As observed in Figure 5B2, the small differences in the counts between the manual method vs. the old macro
and between manual counts vs. the new procedure were similar in low-complexity images.
However, in high-complexity images (Figure 5B1), larger count differences were observed, although those between the manual counts
vs. the new procedure were much lower than those between the manual counts vs. the
old macro.

However, when images were grouped by background and complexity (Figure 6), the differences increased more with the complexity of the DIs than with the presence
of the background. High-complexity DIs with a background (Figure 6A3) gave the greatest differences between manual counts vs. old macro (red curve), with
those between manual counts and the new procedure being the next biggest (green curve).
High-complexity images without background (Figure 6A4) gave the same difference for the comparison between the old macro and the new procedure
with the manual readings (red and green curves).

Figure 6. Kaplan-Meier curves comparing the probability of difference between the manual reading
1 vs. manual reading 2 (in black), the mean of manual readings vs. old macro (in red)
and mean of manual readings vs. new procedure (in green). The X axis represents count
differences between the two methods or readings compared and the Y axis represents
the probability of observing these differences. DIs have been grouped according to
their complexity and their background level: (A1) low-complexity DIs with background,
(A2) low-complexity DIs without background, (A3) high-complexity DIs with background
and (A4) high-complexity DIs without background.

A general point regarding the proposed method is that, for high-complexity DIs with
background (Figure 6A3), the quantification of nuclear markers obtained with the new procedure (green curve)
was closer to the gold standard (manual method, black curve) than with the old macro
(red curve). For high-complexity DIs without background (Figure 6A4), the two automated methods gave the same results (red and green curves). The presence
or absence of background did not appear have a great influence on the quantification
of nuclear markers in low-complexity DIs (Figure 6A1-A2), probably because these images have a lower background level than the other images.

Conclusions

Despite specific and careful preparation of tissues and the use of blocking buffers,
strong background staining can sometimes mask the detection of the target antigen
during automated analysis. The results of the method presented in this paper are promising
since the selective identification of brown color ranges and morphological parameters
of selected objects in DIs enables the background to be discriminated during the automated
localization and quantification of specific stained nuclei. The principle of this
approach is applicable to all quantitative nuclear signals and should prove useful
in a variety of tumor specimens, irrespective of the immunohistochemical techniques
employed.

List of abbreviations used

Competing interests

The authors declare that they have no competing interests.

Author contributions

ML, CL and JJ conceived the study. CL designed and implemented the algorithms. JB
and AK provided IT support. RB and JJ carried out the manual quantification, discussed
the methods and modified them. VG, BT and CC realized the automated quantification
with the two automated methods and designed the database. AR analyzed the data. ML,
CL and AK drafted the manuscript. All authors revised the manuscript and approved
the final version.